Assay for trichomonas vaginalis by amplification and detection of trichomonas vaginalis ap65-1 gene

ABSTRACT

A region of the  Trichomonas vaginalis  AP65-1 gene has been identified which is useful for performing amplification assays to determine specifically whether  T. vaginalis  is present in the sample being tested. Oligonucleotides useful for performing thermal Strand Displacement Assay (tSDA) reactions on this gene are disclosed. The disclosed oligonucleotides can be used in an assay which is specific for multiple strains of  T. vaginalis  and which does not show cross reactivity with the genomes of other microorganisms or with human DNA.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S.Provisional Patent Application No. 61/205,017 filed Jan. 14, 2009, thedisclosure of which is hereby incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Jan. 12, 2010, is namedBECT242.txt, and is 7,805 bytes in size.

BACKGROUND OF THE INVENTION

Trichomonas vaginalis is an anaerobic, parasitic flagellated protozoanthat is the causative agent of trichomoniasis. It is the most commonpathogenic protozoan infection of humans in industrialized countries. Itis estimated by the World Health Organization (WHO) that 180 millioninfections are acquired annually worldwide. The estimates for NorthAmerica alone are between 5 and 8 million new infections each year, withan estimated rate of asymptomatic cases as high as 50%.

Trichomoniasis is a sexually transmitted disease which can occur inmales and females. Symptoms of T. vaginalis typically experienced inwomen include: Vaginitis—itching, burning, and inflammation of thevagina; Cervicitis—inflammation of the cervix; Urethritis—inflammationof the urethra; or a green/yellow, frothy vaginal discharge. Inaddition, the infection may cause discomfort during intercourse andurination, as well as irritation and itching of the female genital area.In rare cases, lower abdominal pain can occur. Symptoms in women usuallyappear within 5 to 28 days of exposure.

Most men with trichomoniasis do not exhibit signs or symptoms. Althoughrare, some men may temporarily have an irritation inside the penis, milddischarge, or slight burning after urination or ejaculation.

There are several known ways to diagnose and detect Trichomoniasis. Oneclassic form of detection is the pap smear, which displays a transparent“halo” around the superficial cell nucleus from samples of infectedindividuals. However, Trichomoniasis is rarely detected by studyingdischarge or with a pap smear because of the low sensitivity associatedwith this form of detection. T. vaginalis was traditionally diagnosedvia a wet mount, in which “corkscrew” motility was observed. In women, adoctor may collect a specimen during a pelvic examination by inserting aspeculum into the vagina and using a cotton-tipped applicator to collecta sample. The sample is then placed onto a microscopic slide and sent toa laboratory for analysis. However, detection via wet mount is lesssensitive than newer methods such as rapid antigen testing andtranscription-mediated amplification. Huppert et al., CID 2007:45 p.194. These newer methods have greater sensitivity, but are not inwidespread use.

Currently, the most common method of laboratory analysis is viaovernight culture of the T. vaginalis organism. Sood, et al., Indian J.Med. Res. 125, April 2007, pp. 567-571; Ohlemeyer, et al., Journ. Of Ad.Health, 22:3 pp. 205-208 (March 1998). However, the presence of T.vaginalis can also be diagnosed by PCR, using the primers L23861 Fw andRev. Sichirm, et al., Journ. Of Microbiological Methods, Vol. 68:2, pp.243-247, (February 2007).

Thus, new diagnostic techniques aimed at more reliably and accuratelydetecting T. vaginalis are desired.

SUMMARY OF THE INVENTION

The adhesion protein gene (i.e. the AP65-1 gene, SEQ ID NO:1), which isabout 1.7 kb in length, is present in T. vaginalis. The AP65-1 gene hasbeen identified in at least three strains of T. vaginalis (ATCC 30001,ATCC 30238, ATCC 300239). It has been unexpectedly determined that aportion of the AP65-1 gene is highly conserved among these three strainsof T. vaginalis and unique to the T. vaginalis organism. Highlyconserved, as used herein, means that the identified portion isconsidered homologous among at least the three above-identified strains.Specifically, the portion of the AP65-1 gene from about base pairs 317through about 560 is determined to be conserved among the three strainsof T. vaginalis.

Oligonucleotides described herein are used to detect the presence of T.vaginalis using the AP65-1 gene. Specifically, oligonucleotidesdescribed herein select for certain portions of the AP65-1 gene andamplify portions thereof for detection. More specifically, theoligonucleotides described herein are used to select for and amplify oneor more portions of the AP65-1 nucleic acid sequence within the organismas a mechanism to detect T. vaginalis. Even more specifically, theoligonucleotides described herein target the conserved portion (basepairs 317 to 560 of Genbank Accession U18346) of the T. vaginalis AP65-1gene. The target portion of the AP65-1 gene is illustrated in FIGS. 1Aand 1B.

Oligonucleotide probe sets described herein are designed to select forthe AP65-1 gene and offer a mechanism for detection. The probe setdesign is based upon a number of factors, chief among which is the assayin which the probe set is used. Assays for the detection of DNA or RNAsequences are well known in the art. These assays typically use sometype of amplification or some type of imaging to confirm the presence ofthe target DNA. Examples of amplification reactions include PCR(polymerase chain reaction), SDA (strand displacement amplification),TMA (transcription mediated amplification) and LCR (ligase chainreaction).

In one embodiment, the amplification mechanism selected for detection isSDA. SDA is an isothermal amplification mechanism and therefore does notinvolve thermal cycling. As such, SDA probe sets are designed for atarget melting temperature (T_(m)) within a predetermined narrow range.Target melting temperature (T_(m)) is the temperature at which at leastfifty percent of the oligonucleotide is annealed to its perfectcomplement. One skilled in the art is aware that the T_(m) of anoligonucleotide sequence is determined by the number of base pairs inthe sequence as well as the type of bases in the sequence. Theseguidelines for designing oligonucleotides are well know to one skilledin the art and are not set forth in detail herein.

It is advantageous if the target site within the AP65-1 gene used inconjunction with the oligonucleotides described herein does not havelong stretches of repeated bases. That is, no more than 3 or 4 bases inthe sequence are the same base. Minimizing the number of baserepetitions permits a system design with optimal melting temperaturesfor the oligonucleotides. Furthermore, the oligonucleotides that bind tothose discrete portions of the AP65-1 gene will not interact with eachother when placed within close proximity.

Suitable binding sites on the AP65-1 gene for one embodiment of an SDAprobe set are listed in the following Table 1 along with their locationon the conserved portion of the AP65-1 gene.

TABLE 1 SEQUENCE Location* SEQ ID Number TGGTTGACAGCCACTTC 352-368 SEQID NO: 2 TGGCGCCGTAGATG 402-415 SEQ ID NO: 3 CTCTGGCTCTTATG 507-520 SEQID NO: 4 TACAGGACCGCAC 465-477 SEQ ID NO: 5 CCTGTAGGAGGCGTTGATG 445-463SEQ ID NO: 6 *Genbank Accession No. U18346

The oligonucleotide SDA probe sets described herein are sufficientlycomplementary to portions of the AP65-1 gene so that they selectivelybind to those portions.

For the SDA embodiment described herein, the oligonucleotide probe sethas left and right bumper primers, left and right amplification primersand a probe. In a preferred embodiment these primers and probes haveoligonucleotide sequences that are the perfect complement to thesequences described above. Specifically, the left and right bumperprimers have the sequences ACCAACTGTCGGTGAAG (SEQ ID NO:7) andGAGACCGAGAATAC (SEQ ID NO:8) respectively. SEQ ID NO:7 is the perfectcomplement of SEQ ID NO:2 and SEQ ID NO:8 is the perfect complement ofSEQ ID NO:4. The left and right primers contain the respective sequencesACCGCGGCATCTAC (SEQ ID NO:9) and ATGTCCTGGCGTG (SEQ ID NO:10). SEQ IDNO:9 is the perfect complement to SEQ ID NO:3. SEQ ID NO:10 is theperfect complement of SEQ ID NO:5. The SDA probe set also includes anoligonucleotide probe that has a sequence GGACATCCTCCGCAACTAC (SEQ IDNO:11) which is the perfect complement of SEQ ID NO:6. One skilled inthe art will appreciate that less than perfect complementarity isrequired as long as the T_(m) requirements and other assays conditionsare met.

The primers and probe have additional nucleotides attached thereto. Theprobe also has additional imaging moieties affixed thereto. Thesemoieties facilitate the detection of the target DNA sequence. Using thisoligonucleotide probe set, an SDA assay may be performed on a sample inorder to determine the presence or absence of all three strains of T.vaginalis. In one illustrative embodiment, about a 75 base pair regionof the AP65-1 gene is amplified between about base pair 317 and 560.Even more specifically, the 75 base pair region of the AP65-1 gene isamplified between base pairs 402 and 477.

In an alternative embodiment, the amplification mechanism selected fordetection is Taqman® real-time PCR assay. Oligonucleotide sequences bindto the AP65-1 gene region between about base pair 317 to about base pair560. Primer/probe sets are configured to not only selectively bind inthis region of the AP65-1 gene, but to amplify some portion of theAP65-1 gene sequence for detection. The oligonucleotides describedherein have a sequence that is capable of binding to the target nucleicacid sequence (and its complementary strand). The oligonucleotidesdescribed herein may also be used, either alone or in combination, tofacilitate detection through amplification of AP65-1 gene nucleic acidsequence. Examples of three probes sets used for Taqman® real-time PCRassays, described in terms of their oligonucleotide sequences, are:

TABLE 2 Probe description: Oligonucleotide 5′ Sequence 3′ AP65-1 Taqman®GAAGATTCTGGCAAGATCAAGGA Forward Primer 1 (SEQ ID NO: 12) AP65-1 Taqman®ACGACAATGCAGCGGATGT Reverse Primer 1 (SEQ ID NO: 13) AP65-1 gene Taqman®ATCCTCCGCAACTACCCACGCCA Probe 1 (SEQ ID NO: 14) AP65-1 gene Taqman®TTACACACCAACTGTCGGTGAAG Forward Primer 2 (SEQ ID NO: 15) AP65-1 geneTaqman® ATGTAGATGCCGCGGTATGAT Reverse Primer 2 (SEQ ID NO: 16) AP65-1gene Taqman® TTGCCAGAAGTGGGCTACACACACCGTC Probe 2 (SEQ ID NO: 17) AP65-1gene Taqman® CAGAGGAAACAATGCCAATTCTT Forward Primer 3 (SEQ ID NO: 18)AP65-1 gene Taqman® TGACGGTGTGTAGCCCACTTC Reverse Primer 3 (SEQ ID NO:19) AP65-1 gene Taqman® ACACCAACTGTCGGTGAAGCTTGCC Probe 3 (SEQ ID NO:20)

In yet another embodiment, the oligonucleotides may be used in a methodfor detecting the presence or absence of T. vaginalis in a sample. In afurther embodiment, the method includes treating a sample using one ormore oligonucleotides specific for the target sequence in a nucleic acidamplification reaction and detecting the presence or absence of theamplified nucleic acid product.

In one illustrative embodiment SDA is selected as the amplificationreaction. In the context of this embodiment, the oligonucleotidesdescribed herein as suited for use in the SDA assay are used incombination as amplification primers, bumper primers and a detector inthat assay.

In another embodiment, a kit is provided for the detection of T.vaginalis. The kit includes one or more of the oligonucleotidesdescribed herein that selectively bind to the AP65-1 gene of T.vaginalis and are capable of amplifying a target sequence that may beused for detection of that organism. The kit is provided with one ormore of the oligonucleotides and buffer reagents for performingamplification assays.

In one aspect of the kit, oligonucleotides and reagents for purposes ofSDA may be provided. In this aspect, two oligonucleotides are providedas amplification primers, two oligonucleotides are provided as bumperprimers and one oligonucleotide may be provided for use as a detector.

In yet another aspect of the kit, the oligonucleotides for SDA purposesmay be provided in dried or liquid format. In dried format, thecomposition may be applied to an appropriate receptacle where sample andproper SDA buffers may be added to perform the assay.

In yet another aspect of the kit, oligonucleotides and reagents forpurposes of Taqman® PCR may be provided. In this aspect, threeoligonucleotides are provided. Two of the three are amplificationprimers and the third oligonucleotide is configured as a detector.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A schematically illustrates an SDA probe set and the targetbinding sites to which the probes attach in the portion of the AP65-1gene (SEQ ID NO: 24);

FIG. 1B illustrates three Taqman® probe sets and the target bindingsites to which the probes attach in the portion of the AP65-1 gene (SEQID NO: 24).

DETAILED DESCRIPTION OF THE INVENTION

Described herein is a method of detecting Trichomonas vaginalis using anassay that consists of one or more oligonucleotide probes that bind tothe AP65-1 gene. Applicant has determined that a region of the AP65-1gene among at least three strains of T. vaginalis (ATCC 30001, ATCC30238 and ATCC 300239) is highly conserved. That is, a region of theAP65-1 gene is sufficiently homologous among the three T. vaginalisstrains so that the oligonucleotide probes are capable of binding to theidentified region of the AP65-1 gene for all three strains. It is alsocontemplated that the oligonucleotide probes are capable of binding to acorresponding region of the AP65-1 gene in other T. vaginalis strains.

The oligonucleotides described herein bind to a region of the AP65-1gene. Specifically, the oligonucleotides described herein target theregion of base pairs 317 to 560 of the AP65-1 gene. The region of basepairs 317 to 560 of the AP65-1 gene has been observed among threestrains of T. vaginalis (ATCC 30001, ATCC 30238 and ATCC 300239) andidentified as highly conserved among at least these three strains. Theinvention exploits this conserved region and the stability of the AP65-1gene among at least three strains of T. vaginalis.

The AP65-1 gene is believed to be unique to T. vaginalis. Due to theconservation and stability of this gene generally, the invention canminimize or eliminate cross reacting/detecting other organisms. Theconservation and stability between base pairs 317 to 560 of the AP65-1gene advantageously do not appear to demonstrate homology with otherparasitic organisms, which allows use of this region of the AP65-1 genefor specific detection of T. vaginalis and reduces the risk of falsepositives.

The AP65-1 gene belongs to the AP65 (adhesion protein 65) multigenefamily encoding multiple homologous 65-kDa proteins whose proteinsequences are identical to those of the hydrogenosomal malic enzymes.These proteins have also been detected on plasma membranes as part ofadhesion complexes during host-parasite encounters. Without being boundby any theory Applicant believes that the AP65-1 gene encodes proteinsassociated with a receptor on T. vaginalis that allows it to bind atarget site on its host. As such, it believed the AP65-1 gene plays asignificant role in the pathogenicity of T. vaginalis.

Thus, because the AP65-1 gene may play a role in pathogenicity and it isstable/conserved, it is advantageous to use for detection of T.vaginalis. The disclosed oligonucleotides are designed to detect theAP65-1 gene because the likelihood of cross reaction/detection withother organisms or eukaryotic cell that could be found in the patient'ssample is minimal.

The oligonucleotide probes and probes sets described herein arespecifically designed to target the AP65-1 gene nucleic acid, and may beused for detecting T. vaginalis. More specifically, the oligonucleotidestarget the conserved portion of the T. vaginalis AP65-1 gene. Theembodiments described herein provide oligonucleotides that select for anucleic acid sequence in T. vaginalis.

The probe sets provide a detectable signal when the area targeted by theoligonucleotides is present in the sample. This is a highly reliableindication of the presence of the AP65-1 gene and, in turn, is a highlyreliable indication for Trichomonas vaginalis.

In the preferred embodiments, the oligonucleotide probes and probe setsare configured to assay for the AP65-1 gene using DNA sequencedetection. Often times, detection assays involve the use ofamplification or imaging to confirm the presence of DNA. Such reactionsinclude SDA, tSDA or homogeneous real time fluorescent tSDA. Thesemethods are known to those skilled in the art from references such asU.S. Pat. No. 5,547,861 and U.S. Pat. No. 5,648,211, U.S. Pat. No.5,928,869 and U.S. Pat. No. 5,846,726 the disclosures of which arehereby incorporated herein by reference. Other methods such as PCR (e.g.Taqman® PCR), TMA, and LCR may also be used. Further, a kit fordetecting T. vaginalis is disclosed.

The oligonucleotides as described herein target the AP65-1 genecontained within T. vaginalis. The AP65-1 gene is known in the art andits sequence is about 1.7 kb in length. See Genbank Accession NumberU18346.

One such probe set, specifically designed for the SDA assay, ispresented in Table 3 below.

TABLE 3 ORF ~Tm Location* SEQ ID Description Oligonucleotide Sequence5′-3′ (° C.) (bp) SEQ ID NO: 7 Left Bumper ACCAACTGTCGGTGAAG 52 352-368(upstream) SEQ ID NO: 21 Left Primer CGATTCCGCTCCAGACTTCTCGGGACCGCGGCATCTAC 46 402-415 (upstream) SEQ ID NO: 8 Right BumperGAGACCGAGAATAC 42 507-520 (downstream) SEQ ID NO: 22 Right PrimerACCGCATCGAATGACTGTCTCGGG ATGTCCTGGCGTG 42 465-477 (downstream) SEQ IDNO: 23 Probe- (6-Fam)-TCCCCGAG(dT)- 60 445-463 detectorDabCylGGACATCCTCCGCAACTAC *Genbank Accession U18346

The left bumper oligonucleotide (ACCAACTGTCGGTGAAG; SEQ ID NO:7) mayhybridize to a complementary target sequence contained within the AP65-1gene. More specifically, left bumper binds to the location at about352-368 base pairs of the AP65-1 gene. This oligonucleotide sequence wasspecifically designed to bind to this particular region of the AP65-1gene.

The left primer oligonucleotide includes SEQ ID NO:9 (ACCGCGGCATCTAC)and may hybridize to a complementary target sequence contained withinthe AP65-1 gene. More specifically, left primer binds to the location atabout 402-415 base pairs of the AP65-1 gene. The left primer wasspecifically designed to bind to this particular region of the AP65-1gene.

The right bumper oligonucleotide (GAGACCGAGAATAC; SEQ ID NO:8) mayhybridize to a complementary target sequence contained within the AP65-1gene. More specifically the AP65-1 gene right bumper binds to thelocation at about 507-520 base pairs of the AP65-1 gene. Thisoligonucleotide sequence was designed to bind this particular region ofthe AP65-1 gene.

The right primer oligonucleotide contains SEQ ID NO:10 (ATGTCCTGGCGTG)and may hybridize to a complementary target sequence contained withinthe AP65-1 gene. More specifically, the right primer binds to thelocation at about 465-477 base pairs of the AP65-1 gene.

The oligonucleotide probe that contains SEQ ID NO:11(GGACATCCTCCGCAACTAC) was designed to specifically bind to base pairs445-463 of the AP65-1 gene.

The probes described above are described in terms of being 100%complementary to their target binding sequences. As described below,primers and probes can bind to target sequences even though they areless than 100% complementary with those regions. The requisite degree ofcomplementarity depends on a variety of factors including the stringencyof the binding conditions. Depending upon the stringency conditionsemployed, the primers and probes may be modified to include differentbases in their sequence and still be sufficiently complementary to bindto the target region of the AP65-1 nucleic acid. Sufficientlycomplementary, as used herein include complementarity of 70% or more. Inpreferred embodiments, the complementarity of the primers/probes totheir target sequence is at least 80% over the length of the bindingportion of the primers/probes. More preferably, the complementarity ofthe primers and probes to their target sequences is 90% or more.

While the oligonucleotides described herein must be sufficientlycomplementary to bind their respective portions of the AP65-1 nucleicacid, it is recognized at some point the sequence of the oligonucleotidebecomes less complementary to the sequence in the AP65-1 nucleic acidand may bind other nucleic acid sequences. Therefore, it is desirablethat the oligonucleotide probes remain sufficiently complementary withits respective portion of the AP65-1 gene, and not lose selectivity forits respective target binding site.

The oligonucleotide probe set described above is configured for use inSDA. However, it is understood that with routine experimentation, one ofskill in the art may use the oligonucleotide sequences described hereinwith or without modification as probes for use in other assays.

The oligonucleotides described herein may be used to amplify a nucleicacid sequence within the target region of the AP65-1 gene. In addition,any sequence which may be produced as a result of an amplificationreaction, referred to as amplification products, amplimers, oramplicons, may serve as amplifiable target sequence for theoligonucleotides described herein.

In the context of SDA, the oligonucleotide probe set as described mayamplify a 75 base pair portion of the AP65-1 gene. Thus,oligonucleotides may amplify a naturally occurring AP65-1 nucleic acidsequence, the complementary second strand of the naturally occurringAP65-1 gene nucleic acid sequence, and either strand of a copy of thenatural occurring AP65-1 gene sequence, which may be produced as aresult of an amplification reaction.

An amplification primer is generally used for amplifying a targetsequence by extension of the primer after hybridization to the targetsequence. Amplification primers are typically about 10-75 nucleotides inlength, preferably about 15-50 nucleotides in length. The total lengthof an amplification primer for use in SDA is typically about 25-50nucleotides.

An amplification primer of one embodiment of the invention as describedherein is useful for SDA and generally has three types of sequences. Onesequence, a target binding sequence, within the primer may be capable ofbinding or hybridizing to the target sequence. Another sequence withinthe primer may be a recognition site for a restriction endonuclease. Yetanother sequence within the primer may act as a repriming sequence.

The target binding sequence within the oligonucleotide amplificationprimer may generally be located at its 3′ end. The target bindingsequence may be about 10-25 nucleotides in length and may havehybridization specificity to the amplification primer. Thus, it isunderstood that one skilled in the art may change the target bindingsequence to effectively change hybridization specificity of theamplification primer and direct hybridization to an alternativesequence.

An SDA amplification primer may also have a recognition site for arestriction endonuclease 5′ to the target binding sequence. Therecognition site on the amplification primer may allow for a restrictionendonuclease to nick one strand of a DNA duplex when the recognitionsite is hemimodified. This is described by G. Walker, et al. (1992. PNAS89:392-396 and 1992 NucL Acids Res. 20:1691-1696). The nucleotides 5′ tothe restriction endonuclease recognition site (the “tail”) function as apolymerase repriming site when the remainder of the amplification primeris nicked and displaced during SDA.

The repriming function of the tail nucleotides sustains the SDA reactionand may allow synthesis of multiple amplicons from a single targetmolecule. The tail is generally about 10-25 nucleotides in length. Itslength and sequence are generally not critical and can be routinelyselected and modified. As the target binding sequence is the portion ofa primer which determines its target-specificity, for amplificationmethods which do not require specialized sequences at the ends of thetarget the amplification primer generally consists essentially of onlythe target binding sequence. For amplification methods which requirespecialized sequences appended to the target other than the nickablerestriction endonuclease recognition site and the tail for SDA (e.g., anRNA polymerase promoter for 3SR, NASBA or transcription basedamplification), may require specialized sequences to link the targetbinding sequence. This may be accomplished using routine methods forpreparation of oligonucleotides without altering the hybridizationspecificity of the primer.

Table 3 depicts two oligonucleotides useful as amplification primers inthe context of SDA. Those amplification primers, the left primer andright primer, are shown with target binding sequences, underlined. Theseportions may hybridize to a target sequence of the AP65-1 gene. The boldsequences indicate a restriction enzyme site. The sequence without anymarkings may act as the tail region.

A bumper primer or external primer is a primer used to displace primerextension products in isothermal amplification reactions. The bumperprimer may anneal to a target sequence upstream of the amplificationprimer such that extension of the bumper primer may displace thedownstream amplification primer and its extension product. Twooligonucleotides, the left bumper and right bumper, described in Table 3may be useful as bumper primers in the context of SDA. Table 3 alsodescribes the SDA probe/detector. The positions of hybridization of theprobes described in Table 3 to the AP65-1 gene when used in an SDAcontext are depicted in FIG. 1.

The target sequence produced by hybridization of a primer and extensionof the primer by polymerase using the target sequence template may bereferred to as the extension product for purposes of discussion herein.

In one embodiment, an SDA system was designed that comprises directdetection using a linear detector probe format. The SDA amplicon size isapproximately 80 base pairs long and contains no BsoB I sites. Oligo 6.0software was used to ensure that no significant secondary structure orinteractions between oligonucleotides exist that would negatively affectSDA performance. Each of the oligos in the T. vaginalis SDA design waschecked using the NCBI site (Blast) and none showed significant homologywith any relevant organism expected to be isolated from a genitourinaryspecimen. Thus, cross-reactivity is expected to be minimal, if any, withother organisms expected to be isolated from a genitourinary specimen.

It is understood to one skilled in the art that the oligonucleotides asused in amplification assays may be modified to some extent without lossof utility or specificity towards a target sequence in T. vaginalis, forexample, the AP65-1 gene. For example, as is known in the art,hybridization of complementary and partially complementary nucleic acidsequences may be obtained by adjustment of the hybridization conditionsto increase or decrease stringency (i.e., adjustment of hybridizationtemperature or salt content of the buffer). Such minor modifications ofthe disclosed sequences and any necessary adjustments of hybridizationconditions to maintain T. vaginalis-specificity require only routineexperimentation and are within the ordinary skill in the art.

As a general guide in designing oligonucleotides useful as primers, Tmdecreases approximately 1° C.-1.5° C. with every 1% decrease in sequencehomology. Temperature ranges may vary between about 50° C. and 62° C.,but the amplification primers may be designed to be optimal at 52° C.However, temperatures below 50° C. may result in primers lackingspecificity, while temperatures over 62° C. may result in nohybridization. A further consideration when designing amplificationprimers may be the guanine and cytosine content. Generally, the GCcontent for a primer may be about 60-70%, but may also be less and canbe adjusted appropriately by one skilled in the art. The hybridizingregion of the target binding sequence may have a Tm of about 42° C.-48°C. Annealing complementary and partially complementary nucleic acidsequences may be obtained by modifying annealing conditions to increaseor decrease stringency (i.e., adjusting annealing temperature or saltcontent of the buffer). Modifications such as those to the disclosedsequences and any necessary adjustments of annealing conditions tomaintain AP65-1 gene specificity require only routine experimentationand are within the ordinary skill in the art.

The amplification products generated using the inventive primers may bedetected by a characteristic size, for example on polyacrylamide oragarose gels stained with ethidium bromide. Alternatively, amplified T.vaginalis AP65-1 gene target sequence may be detected by means of anassay probe, which is an oligonucleotide tagged with a detectable label.In one embodiment, at least one tagged assay probe may be used fordetection of amplified target sequences by hybridization (a detectorprobe), by hybridization and extension as described by Walker, et al.,Nucl. Acids Rev., supra (a detector primer) or by hybridization,extension and conversion to double stranded form as described in EP 0678 582 (a signal primer). Preferably, the assay probe is selected tohybridize to a sequence in the target which is between the amplificationprimers, i.e., it should be an internal assay probe. Alternatively, anamplification primer sequence or the target binding sequence thereof maybe used as the assay probe.

The detectable label of the assay probe may be a moiety which can bedetected either directly or indirectly as an indication of the presenceof the target nucleic acid. For direct detection of the label, assayprobes may be tagged with a radioisotope and detected by autoradiographyor tagged with a fluorescent moiety and detected by fluorescence as isknown in the art. Alternatively, the assay probes may be indirectlydetected by tagging with a label which requires additional reagents torender it detectable. Indirectly detectable labels include, for example,chemiluminescent agents, enzymes which produce visible reaction productsand ligands (e.g., haptens, antibodies or antigens) which may bedetected by binding to labeled specific binding partners (e.g.,antibodies or antigens/haptens). Ligands are also useful forimmobilizing the ligand-labeled oligonucleotide (the capture probe) on asolid phase to facilitate its detection. Particularly useful labelsinclude biotin (detectable by binding to labeled avidin or streptavidin)and enzymes such as horseradish peroxidase or alkaline phosphatase(detectable by addition of enzyme substrates to produce colored reactionproducts). Methods for adding such labels to or including such labelsin, oligonucleotides are well known in the art and any of these methodsare suitable for use in the invention described herein.

Examples of specific detection methods which may be employed include achemiluminescent method in which amplified products are detected using abiotinylated capture probe and an enzyme-conjugated detector probe asdescribed in U.S. Pat. No. 5,470,723. After hybridization of these twoassay probes to different sites in the assay region of the targetsequence (between the binding sites of two amplification primers), thecomplex may be captured on a streptavidin-coated microtiter plate bymeans of the capture probe, and the chemiluminescent signal is developedand read in a luminometer. As another alternative for detection ofamplification products, a signal primer as described in EP 0 678 582 maybe included in the SDA reaction. In this embodiment, labeled secondaryamplification products are generated during SDA in a targetamplification-dependent manner and may be detected as an indication oftarget amplification by means of the associated label.

Oligonucleotide hybridization may be species-specific. That is,detection, amplification or oligonucleotide hybridization in a speciesof organism or a group of related species may occur without substantialdetection, amplification or oligonucleotide hybridization in otherspecies of the same genus or species of a different genus.Oligonucleotides disclosed herein may be useful for identification ofall three strains of T. vaginalis. This includes strains 286, 272 andIR78.

Other sequences, as required for performance of a selected amplificationreaction, may optionally be added to the target binding sequencesdisclosed herein without altering the species-specificity of theoligonucleotide. By way of example, the T. vaginalis AP65-1-specificamplification primers of the invention may contain a recognition sitefor the restriction endonuclease BsoBI which is nicked during the SDAreaction.

It will be apparent to one skilled in the art that other nickablerestriction endonuclease recognition sites may be substituted for theBsoBI recognition site, including but not limited to those recognitionsites disclosed in EP 0 684 315. Preferably, the recognition site is fora thermophilic restriction endonuclease so that the amplificationreaction may be performed under the conditions of thermophilic SDA(tSDA). Similarly, the tail sequence of the amplification primer (5′ tothe restriction endonuclease recognition site) is generally notcritical, although the restriction site used for SDA and sequences whichwill hybridize either to their own target binding sequence or to theother primers should be avoided.

Some amplification primers for SDA according to the invention thereforeconsist of 3′ target binding sequences, a nickable restrictionendonuclease recognition site 5′ to the target binding sequence and atail sequence about 10-25 nucleotides in length 5′ to the restrictionendonuclease recognition site. The nickable restriction endonucleaserecognition site and the tail sequence are sequences required for theSDA reaction. For other amplification reactions, the amplificationprimers according to the invention may consist of the disclosed targetbinding sequences only (e.g., for PCR) or the target binding sequenceand additional sequences required for the selected amplificationreaction (e.g., sequences required for SDA as described above or apromoter recognized by RNA polymerase for 3SR).

In SDA, the bumper primers are not essential for species-specificity, asthey function to displace the downstream, species-specific amplificationprimers. It is only required that the bumper primers hybridize to thetarget upstream from the amplification primers so that when they areextended they will displace the amplification primer and its extensionproduct. The particular sequence of the bumper primer is thereforegenerally not critical, and may be derived from any upstream (to leftamplification primer) or downstream (to right amplification primer)target sequence which is sufficiently close to the binding site of theamplification primer to allow displacement of the amplification primerextension product upon extension of the bumper primer. Occasionalmismatches with the target in the bumper primer sequence or somecross-hybridization with non-target sequences do not generallynegatively affect amplification efficiency as long as the bumper primerremains capable of hybridizing to the specific target sequence. However,the bumper primers described herein are species-specific for T.vaginalis and may therefore also be used as target binding sequences inamplification primers, if desired.

Amplification reactions employing the primers described herein mayincorporate thymine as taught by Walker, et al., supra, or they maywholly or partially substitute 2′-deoxyuridine 5′-triphosphate for TTPin the reaction to reduce cross-contamination of subsequentamplification reactions, e.g., as taught in EP 0 624 643. dU (uridine)is incorporated into amplification products and can be excised bytreatment with uracil DNA glycosylase (UDG). These basic sites renderthe amplification product not amplifiable in subsequent amplificationreactions. UDG may be inactivated by uracil DNA glycosylase inhibitor(Ugi) prior to performing the subsequent amplification to preventexcision of dU in newly-formed amplification products.

Other systems may be used for performing tSDA using differentcombinations of primers, bumpers and detectors. Such systems are wellknown to one skilled in the art and not discussed in detail herein.

A primer mix may be prepared to contain an upstream primer anddownstream primer. The primer mix also contains the upstream anddownstream bumpers. The primers and bumpers may be used at finalconcentrations of about 0.5 and 0.05 uM, respectively.

Oligonucleotide(s) used to facilitate detection or identification of anucleic acid may be used as an assay probe. For example, in theinvention described herein, assay probes may be used for detection oridentification of T. vaginalis AP65-1 nucleic acids. Detector probes,detector primers, capture probes and signal primers as described beloware examples of assay probes.

The primers and probes are preferably used in a tSDA real timefluorescence energy transfer method. Strand Displacement Amplification(SDA) is an isothermal method of nucleic acid amplification in whichextension of primers, nicking of a hemimodified restriction endonucleaserecognition/cleavage site, displacement of single stranded extensionproducts, annealing of primers to the extension products (or theoriginal target sequence) and subsequent extension of the primers occursconcurrently in the reaction mix. This is in contrast to polymerasechain reaction (PCR), in which the steps of the reaction occur indiscrete phases or cycles as a result of the temperature cyclingcharacteristics of the reaction. SDA is based upon 1) the ability of arestriction endonuclease to nick the unmodified strand of ahemiphosphorothioate form of its double stranded recognition/cleavagesite and 2) the ability of certain polymerases to initiate replicationat the nick and displace the downstream non-template strand. After aninitial incubation at increased temperature (about 95° C.) to denaturedouble stranded target sequences for annealing of the primers,subsequent polymerization and displacement of newly synthesized strandstakes place at a constant temperature.

Production of each new copy of the target sequence consists of fivesteps: 1) binding of amplification primers to an original targetsequence or a displaced single-stranded extension product previouslypolymerized, 2) extension of the primers by a 5′-3′ exonucleasedeficient polymerase incorporating an a-thio deoxynucleosidetriphosphate (a-thio dNTP), 3) nicking of a hemimodified double strandedrestriction site, 4) dissociation of the restriction enzyme from thenick site, and 5) extension from the 3′ end of the nick by the 5′-3′exonuclease deficient polymerase with displacement of the downstreamnewly synthesized strand. Nicking, polymerization and displacement occurconcurrently and continuously at a constant temperature becauseextension from the nick regenerates another nickable restriction site.

When a pair of amplification primers is used, each of which hybridizesto one of the two strands of a double stranded target sequence,amplification is exponential. This is because the sense and antisensestrands serve as templates for the opposite primer in subsequent roundsof amplification. When a single amplification primer is used,amplification is linear because only one strand serves as a template forprimer extension. Examples of restriction endonucleases which nick theirdouble stranded recognition/cleavage sites when an a-thio dNTP isincorporated are HincII, HindIII, AvaI, NciI and Fnu4HI. All of theserestriction endonucleases and others which display the required nickingactivity are suitable for use in conventional SDA. However, they arerelatively thermo labile and lose activity above about 40° C.

Targets for amplification by SDA may be prepared by fragmenting largernucleic acids by restriction with an endonuclease which does not cut thetarget sequence. However, it is generally preferred that target nucleicacids having the selected restriction endonuclease recognition/cleavagesites for nicking in the SDA reaction be generated as described byWalker, et al. (1992, Nuc. Acids Res., supra) and in U.S. Pat. No.5,270,184 (hereby incorporated by reference). Briefly, if the targetsequence is double stranded, four primers are hybridized to it. Two ofthe primers (S1 and S2) are SDA amplification primers and two (B1 andB2) are external or bumper primers. S1 and S2 bind to opposite strandsof double stranded nucleic acids flanking the target sequence. B1 and B2bind to the target sequence 5′ of S1 and S2, respectively. Theexonuclease deficient polymerase is then used to simultaneously extendall four primers in the presence of three deoxynucleoside triphosphatesand at least one modified deoxynucleoside triphosphate (e.g.,2′-deoxyadenosine 5′-O-(1-thiotriphosphate), “dATP a S”). The extensionproducts of S1 and S2 are thereby displaced from the original targetsequence template by extension of B1 and B2. The displaced, singlestranded extension products of the amplification primers serve astargets for binding of the opposite amplification and bumper primer(e.g., the extension product of S1 binds S2 and B2). The next cycle ofextension and displacement results in two double stranded nucleic acidfragments with hemimodified restriction endonucleaserecognition/cleavage sites at each end. These are suitable substratesfor amplification by SDA. As in SDA, the individual steps of the targetgeneration reaction occur concurrently and continuously, generatingtarget sequences with the recognition/cleavage sequences at the endsrequired for nicking by the restriction enzyme in SDA. As all of thecomponents of the SDA reaction are already present in the targetgeneration reaction, target sequences generated automatically andcontinuously enter the SDA cycle and are amplified.

To prevent cross-contamination of one SDA reaction by the amplificationproducts of another, dUTP may be incorporated into SDA-amplified DNA inplace of dTTP without inhibition of the amplification reaction. Theuracil-modified nucleic acids may then be specifically recognized andinactivated by treatment with uracil DNA glycosylase (UDG). Therefore,if dUTP is incorporated into SDA-amplified DNA in a prior reaction, anysubsequent SDA reactions can be treated with UDG prior to amplificationof double stranded targets, and any dU containing DNA from previouslyamplified reactions will be rendered not amplifiable. The target DNA tobe amplified in the subsequent reaction does not contain dU and will notbe affected by the UDG treatment. UDG may then be inhibited by treatmentwith Ugi prior to amplification of the target. Alternatively, UDG may beheat-inactivated. In thermophilic SDA, the higher temperature of thereaction itself (≧50° C.) can be used to concurrently inactivate UDG andamplify the target.

SDA requires a polymerase which lacks 5′-3′ exonuclease activity,initiates polymerization at a single stranded nick in double strandednucleic acids, and displaces the strand downstream of the nick whilegenerating a new complementary strand using the unnicked strand as atemplate. The polymerase must extend by adding nucleotides to a free3′-OH. To optimize the SDA reaction, it is also desirable that thepolymerase be highly processive to maximize the length of targetsequence which can be amplified. Highly processive polymerases arecapable of polymerizing new strands of significant length beforedissociating and terminating synthesis of the extension product.Displacement activity is essential to the amplification reaction, as itmakes the target available for synthesis of additional copies andgenerates the single stranded extension product to which a secondamplification primer may hybridize in exponential amplificationreactions. Nicking activity is also of great importance, as it isnicking which perpetuates the reaction and allows subsequent rounds oftarget amplification to initiate.

Thermophilic SDA is performed essentially as the conventional SDAdescribed by Walker, et al. (1992, PNAS and Nuc. Acids Res., supra),with substitution of the desired thermostable polymerase andthermostable restriction endonuclease. Of course, the temperature of thereaction will be adjusted to the higher temperature suitable for thesubstituted enzymes and the HincII restriction endonucleaserecognition/cleavage site will be replaced by the appropriaterestriction endonuclease recognition/cleavage site for the selectedthermostable endonuclease. Also in contrast to Walker, et al., thepractitioner may include the enzymes in the reaction mixture prior tothe initial denaturation step if they are sufficiently stable at thedenaturation temperature. Preferred restriction endonucleases for use inthermophilic SDA are BsrI, BstNI, BsmAI, BslI and BsoBI (New EnglandBioLabs), and BstOI (Promega). The preferred thermophilic polymerasesare Bca (Panvera) and Bst (New England Biolabs).

Homogeneous real time fluorescent tSDA is a modification of tSDA. Itemploys detector oligonucleotides to produce reduced fluorescencequenching in a target-dependent manner. The detector oligonucleotidescontain a donor/acceptor dye pair linked such that fluorescencequenching occurs in the absence of target. Unfolding or linearization ofan intramolecularly base-paired secondary structure in the detectoroligonucleotide in the presence of the target increases the distancebetween the dyes and reduces fluorescence quenching. Unfolding of thebase-paired secondary structure typically involves intermolecularbase-pairing between the sequence of the secondary structure and acomplementary strand such that the secondary structure is at leastpartially disrupted. It may be fully linearized in the presence of acomplementary strand of sufficient length. In a preferred embodiment, arestriction endonuclease recognition site (RERS) is present between thetwo dyes such that intermolecular base-pairing between the secondarystructure and a complementary strand also renders the RERSdouble-stranded and cleavable or nickable by a restriction endonuclease.Cleavage or nicking by the restriction endonuclease separates the donorand acceptor dyes onto separate nucleic acid fragments, furthercontributing to decreased quenching. In either embodiment, an associatedchange in a fluorescence parameter (e.g., an increase in donorfluorescence intensity, a decrease in acceptor fluorescence intensity ora ratio of fluorescence before and after unfolding) is monitored as anindication of the presence of the target sequence. Monitoring a changein donor fluorescence intensity is preferred, as this change istypically larger than the change in acceptor fluorescence intensity.Other fluorescence parameters such as a change in fluorescence lifetimemay also be monitored.

A detector oligonucleotide for homogeneous real time fluorescent tSDA isan oligonucleotide which comprises a single-stranded 5′ or 3′ sectionwhich hybridizes to the target sequence (the target binding sequence)and an intramolecularly base-paired secondary structure adjacent to thetarget binding sequence. The detector oligonucleotides of the inventionfurther comprise a donor/acceptor dye pair linked to the detectoroligonucleotide such that donor fluorescence is quenched when thesecondary structure is intramolecularly base-paired and unfolding orlinearization of the secondary structure results in a decrease influorescence quenching. Cleavage of an oligonucleotide refers tobreaking the phosphodiester bonds of both strands of a DNA duplex orbreaking the phosphodiester bond of single-stranded DNA. This is incontrast to nicking, which refers to breaking the phosphodiester bond ofonly one of the two strands in a DNA duplex.

The detector oligonucleotides of the invention for homogeneous real timefluorescent tSDA comprise a sequence which forms an intramolecularlybase-paired secondary structure under the selected reaction conditionsfor primer extension or hybridization. The secondary structure ispositioned adjacent to the target binding sequence of the detectoroligonucleotide so that at least a portion of the target bindingsequence forms a single-stranded 3′ or 5′ tail. As used herein, the term“adjacent to the target binding sequence” means that all or part of thetarget binding sequence is left single-stranded in a 5′ or 3′ tail whichis available for hybridization to the target. That is, the secondarystructure does not comprise the entire target binding sequence. Aportion of the target binding sequence may be involved in theintramolecular base-pairing in the secondary structure, it may includeall or part of a first sequence involved in intramolecular base-pairingin the secondary structure, it may include all or part of a firstsequence involved in intramolecular base-pairing in the secondarystructure but preferably does not extend into its complementarysequence. For example, if the secondary structure is a stem-loopstructure (e.g., a “hairpin”) and the target binding sequence of thedetector oligonucleotide is present as a single-stranded 3′ tail, thetarget binding sequence may also extend through all or part of the firstarm of the stem and, optionally, through all or part of the loop.However, the target binding sequence preferably does not extend into thesecond arm of the sequence involved in stem intramolecular base-pairing.That is, it is desirable to avoid having both sequences involved inintramolecular base-pairing in a secondary structure capable ofhybridizing to the target. Mismatches in the intramolecularlybase-paired portion of the detector oligonucleotide secondary structuremay reduce the magnitude of the change in fluorescence in the presenceof target but are acceptable if assay sensitivity is not a concern.Mismatches in the target binding sequence of the single-stranded tailare also acceptable but may similarly reduce assay sensitivity and/orspecificity. However, it is a feature of the invention described hereinthat perfect base-pairing in both the secondary structure and the targetbinding sequence does not compromise the reaction. Perfect matches inthe sequences involved in hybridization improve assay specificitywithout negative effects on reaction kinetics.

When added to the amplification reaction, the detector oligonucleotidesignal primers of the invention are converted to double-stranded form byhybridization and extension of an amplification primer as describedabove.

Strand displacement by the polymerase also unfolds or linearizes thesecondary structure and converts it to double-stranded by synthesis of acomplementary strand. The RERS, if present, also becomes double-strandedand cleavable or nickable by the restriction endonuclease. As thesecondary structure is unfolded or linearized by the strand displacingactivity of the polymerase, the distance between the donor and acceptordye is increased, thereby reducing quenching of donor fluorescence. Theassociated change in fluorescence of either the donor or acceptor dyemay be monitored or detected as an indication of amplification of thetarget sequence. Cleavage or nicking of the RERS generally furtherincreases the magnitude of the change in fluorescence by producing twoseparate fragments of the double-stranded secondary amplificationproduct, each having one of the two dyes linked to it. These fragmentsare free to diffuse in the reaction solution, further increasing thedistance between the dyes of the donor/acceptor pair. An increase indonor fluorescence intensity or a decrease in acceptor fluorescenceintensity may be detected and/or monitored as an indication that targetamplification is occurring or has occurred, but other fluorescenceparameters which are affected by the proximity of the donor/acceptor dyepair may also be monitored. A change in fluorescence intensity of thedonor or acceptor may also be detected as a change in a ratio of donorand/or acceptor fluorescence intensities. For example, a change influorescence intensity may be detected as a) an increase in the ratio ofdonor fluorophore fluorescence after linearizing or unfolding thesecondary structure and donor fluorophore fluorescence in the detectoroligonucleotide prior to linearizing or unfolding, or b) as a decreasein the ratio of acceptor dye fluorescence after linearizing or unfoldingand acceptor dye fluorescence in the detector oligonucleotide prior tolinearizing or unfolding.

The oligonucleotides as described may also be useful in otheramplification assays with or without modification. One of ordinary skillin the art would be capable of adapting the oligonucleotide sequences orportions of the oligonucleotide sequences as described herein for otheramplification assays. For example, the oligonucleotide described hereinmay be useful in PCR, TMA, and LCR with or without modification.

It will be apparent that, in addition to SDA, the detectoroligonucleotides of the invention may be adapted for use as signalprimers in other primer extension amplification methods (e.g., PCR, 3SR,TMA or NASBA). For example, the methods may be adapted for use in PCR byusing PCR amplification primers and a strand displacing DNA polymerasewhich lacks 5′-3′ exonuclease activity (e.g., Sequencing Grade Taq fromPromega or exo.sup.—Vent or exo.sup.—Deep Vent from New England BioLabs)in the PCR. The detector oligonucleotide signal primers hybridize to thetarget downstream from the PCR amplification primers, are displaced andare rendered double-stranded essentially as described for SDA. In PCRany RERS may optionally be selected for use in the detectoroligonucleotide, as there are typically no modified deoxynucleosidetriphosphates present which might induce nicking rather than cleavage ofthe RERS. As thermocycling is a feature of amplification by PCR, therestriction endonuclease is preferably added at low temperature afterthe final cycle of primer annealing and extension for end-pointdetection of amplification. However, a thermophilic restrictionendonuclease which remains active through the high temperature phases ofthe PCR reaction could be present during amplification to provide areal-time assay. As in SDA systems, linearization of the secondarystructure and separation of the dye pair reduces fluorescence quenching,with a change in a fluorescence parameter such as intensity serving asan indication of target amplification.

The change in fluorescence resulting from unfolding or linearizing ofthe detector oligonucleotides may be detected at a selected endpoint inthe reaction. However, because linearized secondary structures areproduced concurrently with hybridization or primer extension, the changein fluorescence may also be monitored as the reaction is occurring,i.e., in “real-time”. This homogeneous, real-time assay format may beused to provide semi quantitative or quantitative information about theinitial amount of target present. For example, the rate at whichfluorescence intensity changes during the unfolding or linearizingreaction (either as part of target amplification or in non-amplificationdetection methods) is an indication of initial target levels. As aresult, when more initial copies of the target sequence are present,donor fluorescence more rapidly reaches a selected threshold value(i.e., shorter time to positivity). The decrease in acceptorfluorescence similarly exhibits a shorter time to positivity, detectedas the time required for reaching a selected minimum value. In addition,the rate of change in fluorescence parameters during the course of thereaction is more rapid in samples containing higher initial amounts oftarget than in samples containing lower initial amounts of target (i.e.,increased slope of the fluorescence curve). These or other measurementsas is known in the art may be made as an indication of the presence oftarget or as an indication of target amplification. The initial amountof target is typically determined by comparison of the experimentalresults to results for known amounts of target.

Assays for the presence of a selected target sequence according to themethods of the invention may be performed in solution or on a solidphase. Real-time or endpoint homogeneous assays in which the detectoroligonucleotide functions as a primer are typically performed insolution. Hybridization assays using the detector oligonucleotides ofthe invention may also be performed in solution (e.g., as homogeneousreal-time assays) but are also particularly well-suited to solid phaseassays for real-time or endpoint detection of target. In a solid phaseassay, detector oligonucleotides may be immobilized on the solid phase(e.g., beads, membranes or the reaction vessel) via internal or terminallabels using methods known in the art. For example, a biotin-labeleddetector oligonucleotide may be immobilized on an avidin-modified solidphase where it will produce a change in fluorescence when exposed to thetarget under appropriate hybridization conditions. Capture of the targetin this manner facilitates separation of the target from the sample andallows removal of substances in the sample which may interfere withdetection of the signal or other aspects of the assay.

For commercial convenience, oligonucleotides useful for specificdetection and identification of T. vaginalis AP65-1 nucleic acids may bepackaged in the form of a kit. Typically, such a kit contains at leastone oligonucleotide described herein. Reagents for performing a nucleicacid amplification reaction may also be included with the T. vaginalisAP65-1-specific oligonucleotides. For example, buffers, otheroligonucleotides, nucleotide triphosphates, enzymes, etc. may beincluded. The components of the kit may be packaged together in a commoncontainer. Optionally instructions may be included that illustrate onedescribed embodiment for performing a specific embodiment of theinventive methods. Other optional components may also be included in thekit, e.g., an oligonucleotide tagged with a label suitable for use as anassay probe, and/or reagents or means for detecting the label.

In one embodiment a kit may include at least one oligonucleotide usefulin the context of SDA. Oligonucleotides described herein may be usefulas amplification primers, bumper primers, or probes.

In another embodiment, the kit may include at least one oligonucleotidedescribed herein and optional components useful in the context of SDA.Such optional components may be buffers, nucleotide triphosphates,enzymes, etc. Optionally, reagents for simultaneously detecting a targetsequence, such as a probe, may be included in the kit. One skilled inthe art would understand how to optimize such a kit for amplificationreactions to detect and identify T. vaginalis utilizing theoligonucleotides described herein.

In yet another embodiment, the kit may be used to detect and diagnosewhether a clinical sample contains T. vaginalis AP65-1 DNA. The clinicalsample may be added to the kit so that a nucleic acid sequence may beamplified and detected using the oligonucleotides described herein.

Furthermore, the kit may include oligonucleotides and reagents for SDAin dried or liquid format. The components of the kit may be more stableand easily manipulated when in dried format. The dried components of thekit may be added or pre-treated to a solid phase such as microtiterplate, microarray, or other appropriate receptacle, where the sample andSDA buffer need only be added. This format facilitates assaying multiplesamples simultaneously and is useful in high-throughput methods. The BDProbeTec™ and Viper™ XTR instruments may be used.

The following Examples illustrate specific embodiments of the inventiondescribed herein. As would be apparent to skilled artisans, variouschanges and modifications are possible, and are contemplated within thescope of the invention described.

EXAMPLES Example 1 Assay Sensitivity

The analytical sensitivity of Trichomonas vaginalis ATCC 30001 wasevaluated using the SDA assay at 10⁴/r×n, 10³/r×n and 10²/r×n containinga Bicine/KOH buffer (pH 8.6) with co-solvents. The SDA system thattargets the conserved region of the AP56-1 gene was tested against fourreplicates of the AP56-1 gene (Table 4). The sensitivity for the T.vaginalis SDA assay can be assumed to be equal to or less than 20 targetcopies per assay.

TABLE 4 Assay Sensitivity Replicate 10⁴/rxn 10³/rxn 10²/rxn 1 PositivePositive Positive 2 Positive Positive Positive 3 Positive PositivePositive 4 Positive Positive Positive

The AP65-1 gene SDA assay targets a 75 by region of the AP65-1 genebetween base pairs 402-477.

Example 2 Taqman® PCR System for Detecting AP65-1

Sets of Probes were designed to perform Taqman® PCR on the AP65-1 gene.Taqman® real-time PCR is a type of quantitative PCR. Taqman® uses afluorogenic probe which is a single stranded oligonucleotide of 20-26nucleotides and is designed to bind only the DNA sequence between thetwo PCR primers. In Taqman®, reporter dyes and quencher dyes areattached to the probe. The probe is annealed to the DNA by alternatingthe temperature to denature and re-anneal the DNA. The Taq polymeraseadds nucleotides to the target DNA and this removes the Taqman® probefrom the template DNA. When the reporter dye is separated from thequencher dye, the reporter dye emits energy which is detectable. Theenergy is quantified by a computer, which provides a signal indicatingthat the target was detected.

To practice Taqman® PCR, two PCR primers with a preferred product sizeof 50-150 base pairs and a probe with a fluorescent reporter orfluorophore (e.g. 6-carboxyfluorescein (FAM) and tetrachlorofluorescin(TET)) and a quencher such as tetramethylrhodamine (TAMRA) covalentlyattached to its 5′ and 3′ ends are used. Suitable fluorescent reportersand fluorophores are well known and not described in detail herein.Three exemplary Taqman® probe sets for use in the highly conservedAP65-1 gene are described in Table 7 below. Each probe set consists of aforward primer (FP), a reverse primer (RP) and a probe (P).

TABLE 7 Examples of Tagman® PCR Probes Sets ORF ~Tm Location SEQ ID NO:Name Description 5′ Sequence 3′ (° C.) (bp) SEQ ID NO: 12 AP65-1 AP65-1GAAGATTCTGGCAAGATCAAGGA 58 448-470 Taqman® Taqman® FP Forward Primer SEQID NO: 13 AP65-1 AP65-1 ACGACAATGCAGCGGATGT 59 497-515 Taqman® Taqman®RP Reverse Primer SEQ ID NO: 14 AP65-1 AP65-1 ATCCTCCGCAACTACCCACGCCA 69472494 ID Taqman® gene P Taqman® Probe SEQ ID NO: 15 AP65-1 AP65-1TTACACACCAACTGTCGGTGAAG 59 369-391 Taqman® gene FP2 Taqman® ForwardPrimer 2 SEQ ID NO: 16 AP65-1 AP65-1 ATGTAGATGCCGCGGTATGAT 58 420-440Taqman® gene RP2 Taqman® Reverse Primer 2 SEQ ID NO: 17 AP65-1 AP65-1TTGCCAGAAGTGGGCTACACACACCGTC 70 393-418 Taqman® gene P2 Taqman® Probe 2SEQ ID NO: 18 AP65-1 AP65-1 CAGAGGAAACAATGCCAATTCTT 58 347-369 Taqman®gene FP3 Taqman® Forward Primer 3 SEQ ID NO: 19 AP65-1 AP65-1TGACGGTGTGTAGCCCACTTC 60 399-419 Taqman® gene RP3 Taqman® Reverse Primer3 SEQ ID NO: 20 AP65-1 AP65-1 ACACCAACTGTCGGTGAAGCTTGCC 68 373-397Taqman® gene P3 Taqman® Probe 3

The probes are designed to anneal to the ORF location in the AP65-1 genethat is noted in the Table. FIG. 2 illustrates the binding sites on theAP65-1 gene for the primers and probes described in Table 7.

In addition to the primers and probes, Taqman® PCR requires reagentsthat are used for regular PCR (e.g. polymerase, free nucleotides) aswell as a real-time PCR machine for analyzing the data. The reagents andequipment are well known to those skilled in the art and are notdiscussed in detail herein.

Although the invention herein has been described with reference toparticular embodiments, it is to be understood that these embodimentsare merely illustrative of the principles and applications of theinvention described herein. It is therefore to be understood thatnumerous modifications may be made to the illustrative embodiments andthat other arrangements may be devised without departing from the spiritand scope of the invention described herein as defined by the appendedclaims.

1. A method for detecting Trichomoniasis comprising: providing abiological sample; contacting the biological sample with a substancecomprising an oligonucleotide probe set that comprises at least oneoligonucleotide probe that is detectably labeled and has a nucleotidesequence length of about 10 to about 50 and at least two oligonucleotideprimers, each of which has a nucleotide sequence length of about 10 toabout 150 under conditions such that the probes and primers anneal toSEQ ID NO:1 at the location between about base pairs 317 to 560 on thegene; amplifying the target sequence between the two primers; anddetecting the label as an indication of the hybridization of the probeset to the target sequence thereby indicating the presence or amount ofTrichomoniasis.
 2. The method of claim 1 wherein the probe set comprisesa probe comprising a sequence selected from the group consisting of SEQID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20 and oligonucleotidesequences that are at least 70% homologous to SEQ ID NO:11, SEQ IDNO:14, SEQ ID NO:17 and SEQ ID NO:20.
 3. The method of claim 1 whereinthe probe set comprises a probe comprising a sequence selected from thegroup consisting of SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ IDNO:20 and oligonucleotide sequences that are at least 80% homologous toSEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20.
 4. The method ofclaim 1 wherein the probe set comprises a probe comprising a sequenceselected from the group consisting of SEQ ID NO:11, SEQ ID NO:14, SEQ IDNO:17, SEQ ID NO:20 and oligonucleotide sequences that are at least 90%homologous to SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20. 5.The method of claim 1 wherein the probe set comprises at least twoprimers comprising sequences selected from the group consisting of SEQID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18 and SEQ ID NO:19, andoligonucleotide sequences that are at least 70% homologous to SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18 and SEQ ID NO:19.
 6. Themethod of claim 1 wherein the probe set comprises at least two primerscomprising sequences selected from the group consisting of SEQ ID NO:7,SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:15, SEQ ID NO:16, SEQ ID NO:18 and SEQ ID NO:19, andoligonucleotide sequences that are at least 80% homologous to SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18 and SEQ ID NO:19.
 7. Themethod of claim 1 wherein the probe set comprises at least two primerscomprising sequences selected from the group consisting of SEQ ID NO:7,SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:15, SEQ ID NO:16, SEQ ID NO:18 and SEQ ID NO:19, andoligonucleotide sequences that are at least 90% homologous to SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18 and SEQ ID NO:19.
 8. Themethod of claim 1 wherein the probe set comprises a probe set ofoligonucleotide sequences comprising SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:21, SEQ ID NO:22 and SEQ ID NO:23.
 9. The method of claim 8 whereinthe nucleic acid amplification reaction is Strand DisplacementAmplification (SDA) reaction.
 10. The method of claim 1 wherein theprobe set is selected from the group consisting of a first probe setcomprising primers having an oligonucleotide sequence comprising SEQ IDNO:12 and SEQ ID NO:13 and probe having an oligonucleotide sequencecomprising SEQ ID No:14, a second probe set comprising primers having anoligonucleotide sequence comprising SEQ ID NO:15 and SEQ ID NO:16 andprobe having an oligonucleotide sequence comprising SEQ ID NO:17 and athird probe set comprising primers having an oligonucleotide sequencecomprising SEQ ID NO:18 and SEQ ID NO:19 and probe having anoligonucleotide sequence comprising SEQ ID NO:20.
 11. The method ofclaim 10 wherein the nucleic acid amplification reaction is polymerasechain reaction (PCR).
 12. A probe set for the detection of the AP65-1gene of Trichomonas vaginalis comprising four primers and one probewherein the four primers and one probe each have an oligonucleotideprimer sequence wherein the four oligonucleotide primer sequencescomprise SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10 and theone probe oligonucleotide sequence comprises SEQ ID NO:11.
 13. The probeset of claim 12 wherein the primer comprising SEQ ID NO:9 is SEQ IDNO:21 and the primer comprising SEQ ID NO:10 is SEQ ID NO:22.
 14. Theprobe set of claim 12 wherein the probe comprising SEQ ID NO:11 furthercomprises a detectable marker.
 15. The probe set of claim 13 wherein theprobe comprising SEQ ID NO:11 is SEQ ID NO:23.
 16. The probe set ofclaim 14 wherein said detectable marker of the probe comprising SEQ IDNO:11 is a fluorescence marker.
 17. A kit comprising: a probe set forthe amplification detection of Trichomonas vaginalis that contains aAP65-1 gene (SEQ ID NO:1) a) one or more primers comprisingoligonucleotide sequences that bind to the highly conserved region ofthe SEQ ID NO:1; b) at least one detector comprising an oligonucleotidesequence that binds to a region of SEQ ID NO:1.
 18. The kit of claim 17wherein the at least one detector comprises a detectable marker.
 19. Thekit of claim 17 wherein the detector comprises an oligonucleotidesequence selected from the group consisting of SEQ ID NO:11, SEQ IDNO:14, SEQ ID NO:17, SEQ ID NO:20 and oligonucleotide sequences that areat least 70% homologous to SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17 andSEQ ID NO:20.
 20. The kit of claim 17 wherein the detector comprises anoligonucleotide sequence selected from the group consisting of SEQ IDNO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20 and oligonucleotidesequences that are at least 80% homologous to SEQ ID NO:11, SEQ IDNO:14, SEQ ID NO:17 and SEQ ID NO:20.
 21. The kit of claim 17 whereinthe detector comprises a oligonucleotide sequence selected from thegroup consisting of SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ IDNO:20 and oligonucleotide sequences that are at least 90% homologous toSEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17 and SEQ ID NO:20.
 22. The kitof claim 17 wherein the one or more primers comprises oligonucleotidesequences selected from the group consisting of SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:18 and SEQ ID NO:19, and oligonucleotidesequences that are at least 70% homologous to SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQID NO:16, SEQ ID NO:18 and SEQ ID NO:19.
 23. The kit of claim 17 whereinthe one or more primers comprises oligonucleotide sequences selectedfrom the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:18 and SEQ ID NO:19 and oligonucleotide sequences that are at least80% homologous to SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18 andSEQ ID NO:19.
 24. The kit of claim 17 wherein the one or more primerscomprises oligonucleotide sequences selected from the group consistingof SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12,SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18 and SEQ ID NO:19and oligonucleotide sequences that are at least 90% homologous to SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18 and SEQ ID NO:19. 25.The kit of claim 1 wherein the probe set comprises SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23.
 26. The kit of claim25 wherein the probe set nucleic acid amplification reaction is StrandDisplacement Amplification (SDA) reaction.
 27. The kit of claim 17wherein the probe set is selected from the group consisting of a firstprobe set comprising primers having an oligonucleotide sequencecomprising SEQ ID NO:12 and SEQ ID NO:13 and probe having anoligonucleotide sequence comprising SEQ ID NO:14, a second probe setcomprising primers having an oligonucleotide sequence comprising SEQ IDNO:15 and SEQ ID NO:16 and probe having an oligonucleotide sequencecomprising SEQ ID NO:17 and a third probe set comprising primers havingan oligonucleotide sequence comprising SEQ ID NO:18 and SEQ ID NO:19 andprobe having an oligonucleotide sequence comprising SEQ ID NO:20. 28.The kit of claim 27 wherein the nucleic acid amplification reaction ispolymerase chain reaction (PCR).